The present invention is concerned with the process of blood coagulation which involves an extremely complicated series of interactions generally known as the coagulation cascade, and which is well described in Thrombosis and Hemorrhage, Sec. Ed., 1998, published by Williams and Wilkins. It involves a series of sequential proteolytic actions directed towards coagulation and a complementary series of inhibitory actions involved with termination or inhibition of coagulation. For a better understanding of the invention FIG. 1 displays a part of the blood coagulation cascade. Activation of coagulation takes place along different pathways two of which: the Intrinsic pathway and the Extrinsic pathway are illustrated. These converge to form a common pathway leading to clot formation. Coagulation factors are inactive zymogens or inactive cofactors which, when cleaved by an active protease, are activated (as indicated by the subscript “a”) and in turn activate the next zymogen or the precursor of a cofactor in the cascade.
In the extrinsic pathway, damaged tissue exposes Tissue factor which activates factor VII to its activated form VIIa. Tissue factor and factor VIIa form a complex which activates factor X at the common pathway.
In the intrinsic pathway, negatively charged surfaces are exposed to the action of factor XII and prekallikrein in the bloodstream. Factor XII is activated to factor XIIa which activates factor XI to factor XIa. Factor XIa activates factor IX to IXa. Factor IXa, Factor VIIIa, phospholipids and free calcium ions are required for the formation of the tenase complex, which activates factor X.
Factor Xa, factor Va, phospholipids and free calcium ions are required for the formation of the prothrombinase complex with which this invention is more particularly concerned, which activates prothrombin to thrombin.
Thrombin is the “key enzyme” of coagulation and is linked to many positive and negative feedback actions and also with the clotting process of blood itself. Positive feedbacks include: activation of factors V, VIII and XI. Negative feedbacks include: activation of protein C in the presence of thrombomodulin. The clotting process includes the cleavage of fibrinogen to fibrin and the activation of platelets.
A number of endogenous inhibitory interactions are important, and are presented in the Figure in italics and broken lines. Thus antithrombin, an inhibitor with a relatively broad spectrum whose activity is greatly enhanced by heparins, heparinoids and glycosaminoglycans, inhibits factor Xa, thrombin, factor XIa and factor XIIa. Heparin cofactor II is a more specific endogenous inhibitor which binds to thrombin and whose activity is also accelerated by heparin and additionally by dermatan sulfate. Activated Protein C inactivates factor Villa and factor Va. Tissue factor pathway inhibitor (TFPI) inhibits factor Xa and the tissue factor/factor VIIa complex in a factor Xa dependent fashion.
An appropriate equilibrium of activating and inhibiting factors is necessary for the physiological function of the coagulation system. In certain situations, e.g. hemophilia or lupus, essential factors are missing or materials (anticoagulants) are present which interfere with the coagulation system.
Also substances from animal origin can interfere with coagulation factors. For example hirudin, a protein from the salivary gland of the medicinal leech is a very potent thrombin inhibitor. Proteins from snake venoms can simulate certain factors and lead to coagulation and clotting. In particular some snake venoms activate factor X and/or factor V and can be used in in vitro assays for anticoagulants.
The dependence of the formation of the prothrombinase and tenase complexes on the presence of free calcium ions allows the temporary anticoagulation, e.g. with citrate (or other ionic complexing agent), of a blood sample. This allows the transport and centrifugation of a sample without clotting and also allows the performance of certain analytic reactions without the formation of said complexes.
Subsequent addition of calcium ions can immediately stimulate the activation of prothrombin to thrombin in the presence of phospholipids.
It is often necessary to treat blood with substances having a coagulating (in case of bleeding complications) or anticoagulating effect (for the prevention or treatment of thrombotic complications and during interventions which bring blood into contact with artificial materials, e.g. during extracorporeal blood circulation).
A successful long-term anticoagulation is possible with vitamin K antagonists such as warfarin or other coumarin-derived oral anticoagulants. These substances lead to impaired coagulation activity of the blood by interfering with the formation of certain coagulation factors. As they do not inhibit already formed coagulation factors they need several days, however, until a stable anticoagulation is achieved. In many situations rapid anticoagulation is necessary. One strategy for attaining anticoagulation in patients is the direct or indirect inhibition of activated coagulation factors. The inhibition of certain factors can be achieved using heparins and heparinoids, which require antithrombin and/or heparin cofactor II from plasma, and direct natural or synthetic inhibitors of factor IIa (thrombin) and Xa (e.g. hirudin, argatroban, tick anticoagulant). All these substances mainly target activated factor X (FXa) and/or thrombin. It is important to use an appropriate intensity of anticoagulation since both too high as well as inappropriately low anticoagulation might cause a loss of organ tissue or even death of the patient.
Some anticoagulant drugs e.g. unfractionated heparin (UFH) possess highly variable pharmacokinetics and their use necessitates monitoring of the patient's condition by assaying plasma from the treated patient and, where necessary, adaptation of the individual anticoagulant dosage.
Other anticoagulant strategies such as use of the low molecular weight heparins (LMWH) do not routinely require monitoring of the anticoagulant effect, as the pharmacokinetics are usually less variable. LMWH, containing only part of the glycosaminoglycan heparin chain, is less active than UFH and often considered safer to use. Also with LMWH there are cases where the laboratory assessment of the drug effect is mandatory, e.g. bleeding complications under anticoagulant treatment, suspected or manifestly impaired clearance of the drug (e.g. due to renal dysfunction), unusual pharmocokinetics (e.g. in children or strongly obese patients) or suspected or potential under- and over-dosage. Prolongation of coagulation is dependent on the concentration of anticoagulant in the sample.
The European Pharmacopeial Commission has adopted a standard potency evaluation for LMWH in terms of anti-factor Xa activity (aXa).
Known Coagulation Assays
Methods for measuring the effect on coagulation and/or the concentration in blood or plasma of direct or indirect inhibitors of activated coagulation factors include:
(a) the assessment of inhibition of coagulation factors (e.g. FIIa and FXa) using chromogenic substrate analysis and
(b) so-called “clotting methods”, e.g. the aPTT assay (activated partial thromboplastin time), the ACT assay (activated clotting time), the TT assay (thrombin time), the ECT assay (ecarin clotting time) and the Heptest® assay. The clotting methods are characterised by the fact that coagulation is activated by different regimens and the time from coagulation activation until detection of clotting in the sample is measured. The clotting time can be converted into direct concentration units by establishing a calibration curve with appropriate calibrating reagents.Analysis of Anti-Factor Xa and Anti-Factor IIa Using Chromogenic Substrates
Usually a plasma sample is added to a reagent containing a defined amount of factor IIa or factor Xa (in certain assays antithrombin is also added). During an incubation period factor Xa/IIa is partly inactivated by the anticoagulant itself or by complexes of the anticoagulant with endogenous or exogenous antithrombin. A chromogenic substrate is added and degraded by residual factor Xa/IIa, enhancing optical density which is detected optically. Using a calibration curve the anti-factor Xa/IIa activity or the anticoagulant concentration is calculated.
These methods allow a specific assessment of anticoagulant concentration. However, they are relatively expensive and require specialised instrumentation and are therefore not widely applied in clinical practice. Moreover, the interaction of the anticoagulant with the patient's coagulation system is not assessed and the in vivo situation is not directly reflected.
The aPTT Assay
A blood or (more usually) a plasma sample is added to a reagent containing a contact activator (often substances with negatively charged surfaces like ellagic acid, celite or kaolin) and phospholipids, and is incubated for 2–10 minutes in the absence of calcium ions. The time recorded from the addition of Ca2+ to the sample until detection of fibrin formation is the activated partial thromboplastin time (aPTT).
The aPTT assay has the advantages that it is a widely available test, the clotting method is simple and a large experience base for the monitoring of anticoagulant therapy exists. Although the aPTT is a relatively poorly standardised method, it is frequently employed for the monitoring of unfractionated heparin (UFH), whereas LMWH cannot be monitored with this assay due to its poor responsiveness. In addition to its low sensitivity to LMWH, the assay suffers from a non-linear dose-response relationship to direct thrombin inhibitors like hirudin, too high a sensitivity to UFH, poor standardisation among different instruments, reagents, even different lots of the same reagent. Also the dose response curve for heparin is not linear.
Regarding the mechanism of the aPTT assay, it must be stated that the initiation of coagulation by contact activation (the so-called contact phase) is not part of the physiological hemostatic pathway in the body. The contact phase is difficult to standardise, which is one of the reasons for the very poor standardisation of the assay. Many factors take part in the coagulation activation in the aPTT assay (XII, XI, VIII, IX, X, V, II) while inhibition of factors X and II is believed to be the main pathway of anticoagulant therapy using heparins, heparinoids and the direct inhibitors. For these reasons, the aPTT assay neither gives a very realistic estimation of the anticoagulation achieved in the patient, nor assesses anticoagulation with an appropriate specificity to the anticoagulant treatment. In addition, the assay is also sensitive to the presence of lupus anticoagulants.
The ECT Assay
The Ecarin clotting time assay is a clot based assay used for monitoring the effect of direct antithrombin agents. Ecarin, a purified protease obtained from the venom of the snake Echis carinatus, converts prothrombin to meizothrombin (a precursor of thrombin), producing a clotting end point in citrated whole blood and plasma. Antithrombin agents such as hirudin bind to meizothrombin prolonging the Ecarin clotting time. The ECT assay is highly affected by low prothrombin levels of the sample plasma.
The ACT Assay
This method consists in principle in the addition of blood to kaolin or celite, and measurement of the time interval until fibrin formation in the sample. This method is widely available. It is a point-of-care method with a short turnaround time and a broad measuring range which allows monitoring of high-dose heparinisation during cardiovascular surgery.
The ACT assay has many limitations: it has a poor correlation to the anticoagulant concentration (as assessed by chromogenic substrate analysis), low sensitivity to lower heparin concentrations (up to 0.7 U UFH/ml with a normal ACT), low sensitivity to LMWH, long clotting times and a very strong dependence on the patient's coagulation factors. There is poor standardisation of different clinically applied ACT methods.
The Thrombin Time Assay
The method consists in the addition of a certain amount of thrombin to the plasma sample and assessment of the time interval until clotting is detected. The method has the advantage of specifically assessing thrombin inhibition. Although this simple test is a direct measure for the anti-thrombin activity in plasma, it is not widely used due to its poor reliability and bad standardisation. In addition, no assessment of factor Xa inhibition is possible. The narrow measuring range is strongly dependent on the added thrombin concentration and different results can be obtained in response to thrombin concentration, species, presence of calcium and the volume ratio of thrombin and sample.
The Heparin Assay According to Yin (Presented in 1973)
The assay is based in principle on the incubation of a citrated plasma sample with bovine factor Xa. After a certain incubation time, a reagent containing phospholipids and bovine plasma is added followed by a calcium chloride solution for re-calcification.
This test allows a sensitive assessment of LMWH and UFH by a clotting method. However, it involves a relatively complicated three-step procedure and requires bovine plasma (which might limit the realistic estimation of the anticoagulant effect).
Heptest® Assay (Variation of the Yin Assay (Presented in 1987)
This assay, described in U.S. Pat. No. 4,946,775, consists in principle in the incubation of a sample of plasma or blood with factor Xa. After a certain incubation period, a reagent containing calcium chloride, phospholipids and a bovine plasma fraction is added and the time until detection of clotting is recorded. The bovine plasma fraction is reported by the manufacturer to be rich in factor V and fibrinogen, while it is depleted of prothrombin and other coagulation factors and thus will not clot by itself.
Like the original assay of Yin, the method provides a sensitive assessment of LMWH and UFH in a relatively simple clotting assay. However the Heptest® has a low sensitivity to direct thrombin inhibitors such as hirudin; high standardisation of the bovine plasma fraction is mandatory; the effect of the bovine plasma fraction on the patient's coagulation system is not absolutely defined and its performance on optical analysers can be a problem as the optical signal is not conclusive, as will become apparent. Although the Heptest® is a simple method it is not very widely used.
The chemistry and pharmacology of heparin and assays such as those above outlined are described in Thrombosis and Hemorrhage (op. cit.), and Kandrotas, R. J., Heparin Pharmokinetics and Pharmacodynamics, Clin. Pharmacokinet., vol. 22, 1992, pages 359–374.
Although most known assays are carried out using liquid reagents, systems have been developed, generally for point-of-care application, in which the reagents are supported in a dry state. Examples of such systems are described in U.S. Pat. Nos. 4,756,884, 4,861,712, 5,059,525, 5,110,727 and 5,300,779 and EP-A-680727.
The present invention is aimed at providing a simple and reliable hematological assay which is both sensitive and adjustable in sensitivity to cover the monitoring of a variety of anticoagulants, notably LMWH, UFH, heparinoids, dermatan sulphate, natural or synthetic inhibitors of factor Xa and inhibitors of factor IIa such as argatroban or hirudin, and in its preferred form provides a stable base line when used with optical coagulometers. The invention has many applications in addition to the monitoring of anticoagulant treatment as will become apparent.
The following definitions are used hereafter:
Blood Coagulation Potential
Generally speaking the blood coagulation potential represents the ability of a patient's blood to coagulate or more specifically its ability to activate or inhibit coagulation factors. This is defined for convenience in this specification as a value, which may be given in terms of comparison with, or ratio to, a normal value or standard, of the ability of a sample, e.g. of human whole blood or plasma, or of other mammalian body fluid containing whole blood or plasma, to coagulate to the point of thrombin formation or clotting. The value may be measured in terms of the time taken from induction of coagulation e.g. by the addition to a sample of one or more coagulation accelerants such as phospholipids and calcium ions. However a value indicative of the coagulation potential can be (or can be inferred from) an indirectly measured value, e.g. an indicator value from an added analytical accessory agent, e.g. a chromogenic substrate. This may give a value e.g. for the activity of a component such as factor Xa (which activates prothrombin to thrombin) or of the activity of thrombin.
Coagulation Accelerant
This is defined as a material or substance or mixture of such which greatly speeds up the rate of thrombin formation. It is preferably a substance which completes the group of substances necessary to establish the prothrombinase complex. Thus the fully assembled prothrombinase complex catalyses thrombin formation at a rate that is 300,000 times more efficient than factor Xa acting alone. In addition to factors Va and Xa the prothrombinase complex requires the presence of phospholipid (or platelets) and calcium ions (although other ions can be substituted.)
Analytical Accessory Agent
This is defined as an agent added to a reaction system, e.g. to a sample prior to or, more normally, following treatment to enable the provision of a conveniently observable or otherwise detectable activity. An accessory agent commonly used is a chromogenic substrate: a peptide with distinctive coloured groups which are released when the substrate is acted on by e.g. factor Xa and/or thrombin. Such agents can be specifically designed either for the detection of factor Xa activity or for the detection of thrombin formation. The use of peptide substrates is discussed in Witt, Irene, Test Systems with Synthetic Peptide Substrates in Haemostaseology, Eur. J. Clin. Chem. Clin. Biochem., vol. 29, 1991, pages 355–374.